Laboratory of Plasma Membrane and Nuclear Signaling, Graduate School of Biostudies, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
RIKEN Quantitative Biology Center (QBiC), Suita, Osaka, 565-0874, Japan.
Sci Rep. 2017 Jul 18;7(1):5709. doi: 10.1038/s41598-017-05959-w.
The central channel of the nuclear pore complex (NPC) is occupied by non-structured polypeptides with a high content of Phe-Gly (FG) motifs. This protein-rich environment functions as an entropic barrier that prevents the passage of molecules, as well as the binding sites for karyopherins, to regulate macromolecular traffic between the nucleoplasm and the cytoplasm. In this study, we expressed individual Nups fused with a crowding-sensitive probe (GimRET) to determine the spatial distribution of protein-rich domains within the central channel in vivo, and characterize the properties of the entropic barrier. Analyses of the probe signal revealed that the central channel contains two protein-rich domains at both the nucleoplasmic and cytoplasmic peripheries, and a less-crowded central cavity. Karyopherins and other soluble proteins are not the constituents of the protein-rich domains. The time-lapse observation of the post-mitotic reassembly process also revealed how individual protein-rich domains are constructed by a sequential assembly of nucleoporins.
核孔复合体（NPC）的中央通道被富含苯丙氨酸-甘氨酸（FG）基序的无结构多肽所占据。这种富含蛋白质的环境充当一种熵屏障，防止分子以及亲核蛋白的结合位点通过，从而调节核质和细胞质之间的大分子运输。在这项研究中，我们表达了与拥挤敏感探针（GimRET）融合的单个核孔蛋白，以确定活体内中央通道中富含蛋白质的结构域的空间分布，并对熵屏障的特性进行了表征。探针信号的分析表明，中央通道在核质和细胞质的外周都包含两个富含蛋白质的结构域，以及一个拥挤程度较低的中央腔。亲核蛋白和其他可溶性蛋白不是富含蛋白质的结构域的组成部分。有丝分裂后重新组装过程的时程观察还揭示了单个富含蛋白质的结构域如何通过核孔蛋白的顺序组装来构建。
